Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates which are naturally produced by a large variety of bacteria and fungi. PHAs are biodegradable and renewable, thereby providing an attractive alternative to petroleum-based plastics. However, high production cost has limited the widespread use of PHAs derived from bacterial fermentation. One alternative to reduce cost, production of PHAs in agricultural crops, has been regarded as promising. Small amounts of the PHA have been produced in the cytosol, plastids and peroxisomes of genetically engineered plants. See Poirier (1999) Curr. Opin. Biotechnol. 10(2):181-5; Madison et al. (1999) Microbiol. Mol. Biol. Rev. 63(1):21-53).
PHA synthases catalyze polymerization of hydroxyacyl-CoA substrates into PHA. The substrate specificity of this class of enzymes varies across the spectrum of PHA-producing organisms. The variation in substrate specificity of PHA synthases is supported by indirect evidence observed in heterologous expression studies (Lee et al. (1995) Appl. Microbiol. Biotechnol. 42:901 and Timm et al. (1990) Appl. Microbiol. Biotech. 33:296).
Until recently, the only PHA that has been produced in plants was polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyric acid (John et al. (1996) Proc. Natl. Acad. Sci. USA 93:12768-12773; Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764; Padgette et al. (1997) Plant Physiol. 114 (Suppl.) 3S; Poirier et al. (1992) Science 256:520-523)). Because this polymer is crystalline and brittle with a melting point too close to its degradation point, PHB is difficult to mold into desirable products (Lee (1996) Biotechnol. Bioeng 491.1-14).
Many bacteria make copolymers of 3-hydroxyalkanoic acids with a carbon chain length greater than or equal to five (Steinbuchel (1991) Biomaterials: Novel Materials from Biological Materials, ed. Byrom (New York: Macmillan Publishers Ltd.), pp. 123-213). Such copolymers are polyesters composed of different 3-hydroxyalkanoic acid monomers. Depending on the composition, these copolymers can have properties ranging from firm to elastic (Anderson et al. (1990) Microbiol. Rev. 54:450-472; Lee, (1996) Biotechnol. Bioeng. 49:1-14). Unlike the homopolymeric PHB, the PHA copolymers are suitable for a variety of applications because these copolymers exhibit a wide range of physical properties.
Initial attempts at producing PHA in plants involved producing PHA in the cytosol, but production of PHA in this cellular compartment proved toxic to the plant (Poirier et al. (1992) Science 256:520-523). This problem was overcome by targeting the PHA-producing enzymes to plastids (Nawrath et al. (1994) Proc. Natl. Acad Sci. USA 91:12760-12764). In either cellular compartment, however, only PHB was accumulated, not any of the copolymers. With both of these methods, the genes from Ralstonia eutropha were used. The PHA synthase of this bacterium can utilize only short chain (C3-C5) monomers (Steinbuchel (1991) Biomaterials: Novel Materials from Biological Materials, ed. Byrom (New York: Macmillan Publishers Ltd.), pp. 123-213). Later, copolymer production in Arabidopsis and canola was reported by Slater et al. (1999) Nature Biotechnology 17: 1011-1016.
The synthesis of PHA containing 3-hydroxyalkanoic acid monomers ranging from six to sixteen carbons in Arabidopsis thaliana was reported (Mittendorf et al. (1998) Proc. Natl. Acad. Sci. USA 95:13397-13402). To accumulate PHA, the Arabidopsis plants were transformed with a nucleotide sequence encoding PHA synthase from Pseudomonas aeruginosa that was modified for peroxisome targeting by the addition of a nucleotide sequence encoding the C-terminal 34 amino acids of a Brassica napus isocitrate lyase. In these plants, PHA was produced in glyoxysomes, leaf-type peroxisomes, and vacuoles. However, PHA production was very low in the Arabidopsis plants, suggesting that either the introduced PHA synthase did not function properly in the intended organelle, or more likely that the necessary substrates for the introduced PHA synthase were present at levels that were limiting for PHA synthesis. While this report demonstrated that PHA can be produced in peroxisomes of plants, the level of PHA produced in the plants was far below levels necessary for the commercial production of PHA in plants. Thus, methods and compositions directed to increasing the level of substrate for PHA synthases are needed for production of PHA in plants.
There are two types of fatty acid synthase (FAS). In type I FAS, various enzyme activities are located on different domains of a multifunctional protein. In type II FAS, these enzyme activities are catalyzed by individual polypeptides. 3-oxoacyl-[acyl carrier protein(ACP)] reductase (OAR) is a component of the type II FAS. This enzyme reversibly reduces β-ketoacyl-ACP, the condensation product of an acetyl residue and a nascent acyl-ACP, to β-hydroxyacyl-ACP. In vitro, OAR also uses 3-ketoacyl-CoA as a substrate to catalyze formation of 3-hydroxyacyl-CoA. This use of 3-ketoacyl-CoA is at a lower efficiency than the use of β-ketoacyl-ACP as substrate (Shimakata et al. (1982) Arch. Biochem. Biophys. 218(1): 77-91).
NADPH-dependent OAR from Spinacia oleracea has been described to catalyze the forward reaction of reducing β-ketoacyl-ACP, more than seventeen times faster than the reverse dehydrogenation reaction, at neutral or acidic pH. This OAR has also been shown to use only D-3-hydroxybutyryl-ACP as a substrate but not the L-form counterpart.
NADH-dependent forms of OARs have been described from plant species such as castor bean and avocado (Shimakata et al. (1982) Arch. Biochem. Biophys. 218(1): 77-91; Caughey et al. (1982) Eur. J Biochem. 123(3): 553-61). Taguchi et al. have shown that over-expression of a bacterial NADPH-dependent OAR increases D-3-hydroxyacyl-CoA monomer supply for PHA synthase and leads to accumulation of PHAs in E. coli (Taguchi et al. (1999) Fems. Microbiol. Lett. 176(1): 183-190).
Thus, methods and compositions directed to plant OARs are needed for increasing the level of substrate for PHA synthases, and for production of PHA in plants.